Amino-Acid-Incorporating Nonionic Surfactants for Stabilization of Protein Pharmaceuticals.

With the rapid development of protein-based pharmaceutical products over the past decade, one of the biggest challenges in product development is maintaining the structural stability of proteins during purification, processing, and storage. In this work, the design of a new class of surfactants, polyether-modified N-acyl amino acids, is presented. One surfactant from this series, containing a phenylalanine moiety, demonstrated remarkable stabilization against aggregation of several model protein drugs. Dynamic light scattering, size exclusion chromatography, and circular dichroism all show the rate of thermally accelerated protein aggregation slowed. IgG aggregation was reduced by 3-fold compared to polysorbate controls. Testing of Orencia, a prescription biologic drug for rheumatoid arthritis, demonstrated a 36% improvement in monomer retention upon heat-aging.

2DLC-UV/MS assay for the simultaneous quantification of intact soybean allergens Gly m 4 and hydrophobic protein from soybean (HPS).

Top-down approaches for quantification of proteins based on separation and mass spectrometric assays hold promise due to their high specificity and avoidance of both proteolytic steps and need for generation of monoclonal antibodies. In this study, a 2DLC-UV/MS assay was developed for the simultaneous quantification of two intact soybean allergens, hydrophobic protein from soybean (HPS) and Gly m 4. Both of these allergens were purified from soybean seeds followed by complete characterization. The method validation consisted of evaluating linearity, precision, and recovery. A linear relationship (R(2) > 0.99) between concentrations of the two proteins and their respective peak areas was observed over the concentration ranges from 6.9 to 355.1 µg/mL and from 11.9 to 599.8 µg/mL for Gly m 4 and HPS, respectively. For the 4 day validation study, precision range (%CV) was observed to be from 4.7 to 9.2% for HPS and from 6.3 to 9.4% for Gly m 4. The assay recovery range (%RE) was observed to be from -1.1 to -13.7% for HPS and from -3.5 to 15.2% for Gly m 4. The assay was applied on 10 non-transgenic commercial lines to quantify the relative levels of the two allergens. The HPS and Gly m 4 levels ranged from 64 to 479 µg/g and from 204 to 637 µg/g, respectively. To the best of the authors' knowledge, this represents the first 2DLC-UV/MS assay for the simultaneous quantitation of selected allergens at the intact level.

Characterization of aryloxyalkanoate dioxygenase-12, a nonheme Fe(II)/α-ketoglutarate-dependent dioxygenase, expressed in transgenic soybean and Pseudomonas fluorescens.

Aryloxyalkanoate dioxygenase-12 (AAD-12) was discovered from the soil bacterium Delftia acidovorans MC1 and is a nonheme Fe(II)/α-ketoglutarate-dependent dioxygenase, which can impart herbicide tolerance to transgenic plants by catalyzing the degradation of certain phenoxyacetate, pyridyloxyacetate, and aryloxyphenoxypropionate herbicides. (1) The development of commercial herbicide-tolerant crops, in particular AAD-12-containing soybean, has prompted the need for large quantities of the enzyme for safety testing. To accomplish this, the enzyme was produced in Pseudomonas fluorescens (Pf) and purified to near homogeneity. A small amount of AAD-12 was partially purified from transgenic soybean and through various analytical, biochemical, and in vitro activity analyses demonstrated to be equivalent to the Pf-generated enzyme. Furthermore, results from in vitro kinetic analyses using a variety of plant endogenous compounds revealed activity with trans-cinnamate and indole-3-acetic acid (IAA). The catalytic efficiencies (kcat/Km) of AAD-12 using trans-cinnamate (51.5 M(-1) s(-1)) and IAA (8.2 M(-1) s(-1)) as substrates were very poor when compared to the efficiencies of plant endogenous enzymes. The results suggest that the presence of AAD-12 in transgenic soybean would not likely have an impact on major plant metabolic pathways.

Quantification of Gly m 4 protein, a major soybean allergen, by two-dimensional liquid chromatography with ultraviolet and mass spectrometry detection.

Soybean (Glycine max) is considered a major allergenic food. Gly m 4 is one of several soybean allergens that has been identified to cause an allergic reaction, typically the symptoms are localized effects including the skin, gastrointestinal tract, or respiratory tract. Soybean allergens are considered a complete food allergen in that they are capable of inducing specific IgE as well as eliciting a range of severity from mild rashes up to anaphylaxis. In this study, we have isolated, purified, and characterized an endogenous Gly m 4 protein. The endogenous protein has 88.0% sequence homology with the theoretically predicted Gly m 4 sequence. Following detailed characterization, an assay was developed for quantification of endogenous Gly m 4 using two-dimensional liquid chromatography with ultraviolet and mass spectrometric detection (2DLC-UV/MS). A linear relationship (R(2) > 0.99) was observed over the concentration range of 12.5-531.7 µg/mL. Over the linear range, the assay recoveries (percent relative error, % RE) ranged from -1.5 to 10.8%. The assay precision (percent coefficient of variation, % CV) was measured at three different Gly m 4 levels on each of the 4 days and did not exceed 11.2%. The developed method was successfully applied to quantify Gly m 4 level in 10 commercial soybean lines. To the best of our knowledge, this represents the first quantitative assay for an intact endogenous Gly m 4 protein.

Quantification and characterization of maize lipid transfer protein, a food allergen, by liquid chromatography with ultraviolet and mass spectrometric detection.

Maize (Zea mays) is not considered a major allergenic food; however, when food induced allergenic and immunologic reactions have been implicated to maize, lipid transfer proteins (LTPs) have been identified as major allergens. LTP is an extremely stable protein that is resistant to both proteolytic attack and food processing, which permits the allergen to reach the gastrointestinal immune system in an immunogenic and allergenic conformation, allowing sensitization and induction of systemic symptoms. They are considered a complete food allergen in that they are capable of inducing specific IgE as well as eliciting severe symptoms. We have purified and characterized an endogenous ~9 kDa LTP from maize kernels. The maize LTP consists of 93 amino acid residues and has a M(r) of 9046.1 Da, determined by electrospray ionization mass spectrometry. Following accurate identification and characterization of maize LTP, a highly specific and quantitative assay using liquid chromatography with ultraviolet and mass spectrometric detection was developed. The present assay enables determination of LTP over a concentration range from 29 to 1030 µg/g in maize kernel samples. Assay recovery (percent relative error, % RE) was measured at 11 different concentrations ranging from 4 to 147 µg/mL and did not exceed 5.1%. The precision (percent coefficient of variation, % CV) was measured at 3 concentrations on each of 4 days and did not exceed 14.4%. The method was applied to evaluate the levels of LTP in 14 different maize lines. To our knowledge, this represents the first quantitative liquid chromatography-ultraviolet/mass spectrometry (LC-UV/MS) assay for the determination of LTP for the assessment of a food allergen.

Determination of F2-isoprostanes in urine by online solid phase extraction coupled to liquid chromatography with tandem mass spectrometry.

F(2)-isoprostanes are a unique class of prostaglandin-like compounds formed in vivo, which have been established as biomarkers of oxidative stress. Accurate analysis has been challenging due to lack of specificity for the isoforms of isoprostanes and lengthy sample preparation procedures to enable trace quantitative analysis. A quantitative analytical method was developed for the determination of F(2)-isoprostanes in rat and hamster urine by online solid phase extraction (SPE) coupled with liquid chromatography and tandem mass spectrometry (LC-MS/MS). The online SPE LC-MS/MS procedure has significant advantages over alternative methods with respect to specificity, sensitivity, simplicity, and speed. The assay enables the detection of iPF(2alpha)-III, iPF(2alpha)-IV, and iPF(2alpha)-VI over a linear dynamic range of 0.1-50 ng/mL in rat urine samples. This range covers the basal levels of these F(2)-isoprostanes. The limit of quantitation (LOQ) for the standard isoprostanes was about 0.3 ng/mL. The average recoveries ranged from 73 to 115% depending upon the individual F(2)-isoprostane isomers in rat urine. Additionally, the method was used to determine increases of endogenous urine iPF(2alpha)-VI and iPF(2alpha)-III in hamsters challenged with either low-fat or high-fat diets.

Separation of natural product using columns packed with Fused-Core particles.

Three HPLC columns packed with 3 microm, sub-2 microm, and 2.7 microm Fused-Core (superficially porous) particles were compared in separation performance using two natural product mixtures containing 15 structurally related components. The Ascentis Express C18 column packed with Fused-Core particles showed an 18% increase in column efficiency (theoretical plates), a 76% increase in plate number per meter, a 65% enhancement in separation speed and a 19% increase in back pressure compared to the Atlantis T3 C18 column packed with 3 microm particles. Column lot-to-lot variability for critical pairs in the natural product mixture was observed with both columns, with the Atlantis T3 column exhibiting a higher degree of variability. The Ascentis Express column was also compared with the Acquity BEH column packed with sub-2 microm particles. Although the peak efficiencies obtained by the Ascentis Express column were only about 74% of those obtained by the Acquity BEH column, the 50% lower back pressure and comparable separation speed allowed high-efficiency and high-speed separation to be performed using conventional HPLC instrumentation.

Development of a comprehensive multidimensional liquid chromatography system with tandem mass spectrometry detection for detailed characterization of recombinant proteins.

A multidimensional comprehensive liquid-phase separation system (2DLC) coupled on-line to an electrospray-ionization time-of-flight (ESI-TOF) mass spectrometer (MS) was used to resolve structural alterations and/or post-translational modifications for detailed protein characterization. The system described in this work consists of cation-exchange chromatography in the first dimension and reversed-phase chromatography in the second dimension. A unique spiked gradient was employed in the first dimension to enhance recovery of peptides. This combination of separation followed by MS detection offered the advantages of unique selectivity and high efficiency of the separation methods combined with the mass specificity and sensitivity of MS. During the course of this study it was determined that altered or modified peptides were shown to be better resolved than during a one-dimensional separation. The 2DLC/ESI methodology allowed for a comprehensive evaluation of post-translational modifications and chemical reactions of recombinant proteins, providing a meaningful evaluation of product quality that was not possible with other current analytical approaches. In addition, the system can be used to provide sequence coverage of complex proteins.

A novel HPLC-UV-MS method for quantitative analysis of protein glycosylation.

Monoclonal antibody samples derived from transgenic plants (plantibodies) may often contain significant amounts of aglycosylated variants. Because glycosylated and non-/de-glycosylated proteins exhibit different functional and pharmacokinetic properties, accurate measurement of non- and de-glycosylated glycoprotein abundances is important. Glycosylation of plant-derived glycoproteins presents specific challenges. Here we describe a novel method to accurately measure relative and absolute amounts of non-glycosylated, de-glycosylated, and total glycosylated protein using an HPLC-UV-MS methodology. Additionally, these results were compared with glycopeptide profiling by MALDI MS. Our studies demonstrated that the quantitative aspect of HPLC-UV method was superior to MALDI MS profiling, which significantly overestimated the relative amounts of aglycosylated species in the isolated glycopeptide fractions.

Fragmentation of doubly-protonated peptide ion populations labeled by H/D exchange with CD(3)OD.

Doubly-protonated bradykinin (RPPGFSPFR) and an angiotensin III analogue (RVYIFPF) were subjected to hydrogen/deuterium (H/D) exchange with CD(3)OD in a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. A bimodal distribution of deuterium incorporation was present for bradykinin after H/D exchange for 90 s at a CD(3)OD pressure of 4 x 10(-7) Torr, indicating the existence of at least two distinct populations. Bradykinin ion populations corresponding to 0-2 and 5-11 deuteriums (i.e., D(0), D(1), D(2), D(5), D(6), D(7), D(8), D(9), D(10), and D(11)) were each monoisotopically selected and fragmented via sustained off-resonance irradiation (SORI) collision-induced dissociation (CID). The D(0)-D(2) ion populations, which correspond to the slower exchanging population, consistently require lower SORI amplitude to achieve a similar precursor ion survival yield as the faster-reacting (D(5)-D(11)) populations. These results demonstrate that conformation/protonation motif has an effect on fragmentation efficiency for bradykinin. Also, the partitioning of the deuterium atoms into fragment ions suggests that the C-terminal arginine residue exchanges more rapidly than the N-terminal arginine. Total deuterium incorporation in the b(1)/y(8) and b(2)/y(7) ion pairs matches very closely the theoretical values for all ion populations studied, indicating that the ions of a complementary pair are likely formed during the same fragmentation event, or that no scrambling occurs upon SORI. Deuterium incorporation into the y(1)/a(8) pseudo-ion pair does not closely match the expected theoretical values. The other peptide, doubly-protonated RVYIFPF, has a trimodal distribution of deuterium incorporation upon H/D exchange with CD(3)OD at a pressure of 1 x 10(-7) Torr for 600 s, indicating at least three distinct ion populations. After 90 s of H/D exchange where at least two distinct populations are detected, the D(0)-D(7) ion populations were monoisotopically selected and fragmented via SORI-CID over a range of SORI amplitudes. The precursor ion survival yield as a function of SORI amplitude falls into two distinct behaviors corresponding to slower- and faster-reacting ion populations. The slower-reacting population requires larger SORI amplitudes to achieve the same precursor ion survival yield as the faster exchanging population. Total deuterium incorporation into the y(2)/b(5) ion pairs matches closely the theoretical values over all ion populations and SORI amplitudes studied. This result indicates the y(2) and b(5) ions are likely formed by the same mechanism over the SORI amplitudes studied.